Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′ + 5′ ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription.
Bibliographical noteFunding Information:
We thank Dr. Amnon Hizi for providing purified HIV-1 RT protein and Joy Lengyel and Ming Li for A3G plasmid DNA constructs and sharing unpublished EMSA data. We thank Dr. Cheryl Balshayi for critical reading of this manuscript. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH: anti-APOBEC3G C terminus from Dr. Jaisri Lingappa and anti-APOBEC3G from Dr. Warner C. Greene. This work was carried out in the Peter A. Krueger Laboratory with the generous support of Nancy and Lawrence Glick and Pat and Marvin Weiss. This work was supported by an NIH grant ( P01 GM091743 to R. S. Harris, with a subaward to M.K.) and the United States–Israel Binational Science Foundation . R.N. was a fellow of the Clore Scholars Programme.